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Background: Our study aims to assess the feasibility and the reproducibility of fluorescent confocal microscopy (FCM) real-time assessment of urethral and ureteral margins during open radical cystectomy (ORC) for bladder cancer (BCa). Methods: From May 2020 to January 2022, 46 patients underwent ORC with intraoperative FCM evaluation. Each specimen was intraoperatively stained for histopathological analysis using FCM, analyzed as a frozen section (FSA), and sent for traditional H&E examination. Sensitivity, specificity, positive predictive value (PPV), and the negative predictive value (NPV) of FCM and FSA were assessed and compared with H&E for urethral and ureteral margins separately. Results: The agreement was evaluated through Cohen’s κ statistic. Urethral diagnostic agreement between FCM and FSA showed a κ = 0.776 (p < 0.001), while between FCM and H&E, the agreement was κ = 0.691 (p < 0.001). With regard to ureteral margins, an overall agreement of κ = 0.712 (p < 0.001) between FCM and FSA and of κ = 0.481 (p < 0.001) between FCM and H&E was found. Conclusions: FCM proved to be a safe, feasible, and reproducible method for the intraoperative assessment of urethral and ureteral margins during ORC. Compared to standard FSA, FCM showed adequate diagnostic performance in detecting urethral and ureteral malignant involvement.

Background: Our study aims to assess the feasibility and the reproducibility of fluorescent confocal microscopy (FCM) real-time assessment of urethral and ureteral margins during open radical cystectomy (ORC) for bladder cancer (BCa). Methods: From May 2020 to January 2022, 46 patients underwent ORC with intraoperative FCM evaluation. Each specimen was intraoperatively stained for histopathological analysis using FCM, analyzed as a frozen section (FSA), and sent for traditional H&E examination. Sensitivity, specificity, positive predictive value (PPV), and the negative predictive value (NPV) of FCM and FSA were assessed and compared with H&E for urethral and ureteral margins separately. Results: The agreement was evaluated through Cohen's κ statistic. Urethral diagnostic agreement between FCM and FSA showed a κ = 0.776 (p < 0.001), while between FCM and H&E, the agreement was κ = 0.691 (p < 0.001). With regard to ureteral margins, an overall agreement of κ = 0.712 (p < 0.001) between FCM and FSA and of κ = 0.481 (p < 0.001) between FCM and H&E was found. Conclusions: FCM proved to be a safe, feasible, and reproducible method for the intraoperative assessment of urethral and ureteral margins during ORC. Compared to standard FSA, FCM showed adequate diagnostic performance in detecting urethral and ureteral malignant involvement. [ABSTRACT FROM AUTHOR]

In this study, we aim at demonstrating the possibility for operando quantification of the thickness of the boundary layer where pH changes are occurring upon electrochemical reactions accompanied with the production or consumption of H+ cations. Confocal fluorescent microscopy (CFM) is combined with two different pH sensitive fluorescent dyes, namely 3,4′‐dihydroxy‐3′,5′‐bis‐(dimethylaminomethyl)flavone (FAM345) and rhodamine‐6‐aniline (R6H). This is the first attempt to quantify the effect of the buffer concentration (quantification of layer thickness) on local pH changes produced by ascorbic oxidation and oxygen reduction reaction directly occurring at non‐modified graphite electrodes. In addition, the effect of buffer concentration was studied also with respect to local pH variations produced by bioelectrochemical reactions. [ABSTRACT FROM AUTHOR]

The novel, selective and pH-resistant AIE type chemosensor, based on pyridine, containing a tetracyanobutadiene moiety (4-CN-TCPy) for turn on detection of Ag+, as well as for turn-off detection of I− and mercaptocarboxylic acids was developed. On the example of both tumor and non-tumor cells, it was shown that developed sensor can be used for fluorescent imaging of tissues. [Display omitted] • Highly efficient turn-on AIE chemosensors for detecting of Ag+. • Turn-off chemosensor for detecting of mercaptocarboxylic acids. • Fluorescence imaging of tumor and non-tumor cells. [ABSTRACT FROM AUTHOR]

Topological rearrangements of biological membranes, such as fusion and fission, often require a sophisticated interplay between different proteins and cellular membranes. However, in the case of fusion proteins of enveloped viruses, even one molecule can execute membrane restructurings. Growing evidence indicates that matrix proteins of enveloped viruses can solely trigger the membrane bending required for another crucial step in virogenesis, the budding of progeny virions. For the case of the influenza A virus matrix protein M1, different studies report both in favor and against M1 being able to produce virus-like particles without other viral proteins. Here, we investigated the physicochemical mechanisms of M1 membrane activity on giant unilamellar vesicles of different lipid compositions using fluorescent confocal microscopy. We confirmed that M1 predominantly interacts electrostatically with the membrane, and its ability to deform the lipid bilayer is non-specific and typical for membrane-binding proteins and polypeptides. However, in the case of phase-separating membranes, M1 demonstrates a unique ability to induce macro-phase separation, probably due to the high affinity of M1's amphipathic helices to the raft boundary. Thus, we suggest that M1 is tailored to deform charged membranes with a specific activity in the case of phase-separating membranes.

Reactive oxygen and nitrogen species have been implicated in the loss of skeletal muscle mass and function that occurs during aging. Nitric oxide (NO) and superoxide are generated by skeletal muscle and where these are generated in proximity their chemical reaction to form peroxynitrite can compete with the superoxide dismutation to hydrogen peroxide. Changes in NO availability may therefore theoretically modify superoxide and peroxynitrite activities in tissues, but published data are contradictory regarding aging effects on muscle NO availability. We hypothesised that an age-related increase in NO generation might increase peroxynitrite generation in muscles from old mice, leading to an increased nitration of muscle proteins and decreased superoxide availability. This was examined using fluorescent probes and an isolated fiber preparation to examine NO content and superoxide in the cytosol and mitochondria of muscle fibers from adult and old mice both at rest and following contractile activity. We also examined the 3-nitrotyrosine (3-NT) and peroxiredoxin 5 (Prx5) content of muscles from mice as markers of peroxynitrite activity. Data indicate that a substantial age-related increase in NO levels occurred in muscle fibers during contractile activity and this was associated with an increase in muscle eNOS. Muscle proteins from old mice also showed an increased 3-NT content. Inhibition of NOS indicated that NO decreased superoxide bioavailability in muscle mitochondria, although this effect was not age related. Thus increased NO in muscles of old mice was associated with an increased 3-NT content that may potentially contribute to age-related degenerative changes in skeletal muscle. [ABSTRACT FROM AUTHOR]

Fluorescent microscopy imaging is a popular and well-established method for biomedical research. However, the large number of images created in each research trial quickly eliminates the possibility of a manual annotation; thus, the need for automatic image annotation is quickly becoming an urgent need. Furthermore, the high clustering indexes and noise observed in these images contribute to a complex issue, which has attracted the attention of the scientific community. In this paper, we present a fully automated method for annotating fluorescent confocal microscopy images in highly complex conditions. The proposed method relies on a multi-layered segmentation and declustering process, which begins with an adaptive segmentation step using a two-level Otsu's Method. The second layer is comprised of two probabilistic classifiers, responsible for determining how many components may constitute each segmented region. The first of these employs rule-based reasoning grounded on the decreasing harmonic pattern observed in the region area density function, while the second one consists of a Support Vector Machine trained with features derived from the log likelihood ratio function of Gaussian mixture models of each region. Our results indicate that the proposed method is able to perform the identification and annotation process on par with an expert human subject, thus presenting itself a viable alternative to the traditional manual approach. [ABSTRACT FROM AUTHOR]

Endocytosis of signaling receptors, EGF receptor in particular, starting at the plasma membrane and finishing in perinuclear lysosomes entails endosome multiple interactions with homotypic endosomes and vesicles of other origin (lysosomes, trans-Golgi network), which results in changes of endosome size. A distinctive feature of the endocytic pathway is endosome translocation from the cell periphery to the juxtanuclear region. Thus, endocytosis is a highly dynamic process developing in time and space. One of the most productive approaches to studying endocytosis regulation is light immunofluorescent microscopy, which allows determining the endocytosis dynamics at the level of single or several cells. Different effects that influence endocytic regulator components are inevitably reflected on the dynamics on endosome size and/or its translocation. This makes it possible to reveal both primary and secondary components of the regulatory machinery. However, visual determination of such effects is often subjective and does not allow statistically reliable data to be obtained. Comparison of different experiments, even in the case of the same series, also may be complicated. In this work, we use such parameters as apparent vesicle size (diameter, area, or volume) and vesicle number per cell to provide quantitative estimation of fusion efficacy. Moreover, we propose a coefficient reflecting vesicle clusterization in the perinuclear region as a measure of their translocation along microtubules toward the nucleus ( D). We present the application these parameters using EGF receptor endocytosis as an example. [ABSTRACT FROM AUTHOR]

Abstract: Neutrophil extracellular traps (NETs), which are extracellular DNA structures released from neutrophils, are described and characterized for the first time in fish using fluorescent confocal microscopy. Confocal images of fish neutrophil suspensions stained with 6′-diamino-2-phenylindole, dihydrochloride DNA fluorescent stain (DAPI) revealed the presence of NETs which appeared as fibrous structures connecting several cells. Co-localization of NETs with neutrophil granular proteins and actin was investigated using specific antibodies and probes. Double staining of neutrophils with SYTOX green and DAPI revealed that SYTOX stain applied to living cells stained extracellular DNA, but not nuclei. NETs are actively released from stimulated living cells, associated with granular proteins, but not with cytoskeleton, and are not a product of nuclear degradation seen in late apoptotic stages. Additionally, a fluorometric microtiter plate assay to quantify the release of NETs was adopted for use with fish neutrophils, and the effect of stress on NETs release was studied. This assay detected the inhibition of DNA release during stress conditions. In summary, NETs were released from living fish kidney neutrophils upon stimulation, characterized using fluorescence DNA-binding dyes, specific antibodies and probes, and quantified using a microtiter plate fluorometric assay that can rapidly measure a large number of samples. Detection of NETs can be used as an additional assay to an existing battery of functional tests, and as a new research model to study the effects of stress, immunomodulators, and diseases. [Copyright &y& Elsevier]

A method has been developed for selective detection of the zinc-deficient form of Cu, Zn superoxide dismutase (SOD1) in vitro. Zinc-deficient SOD1 mutants have been implicated in the death of motor neurons leading in amyotrophic lateral sclerosis (ALS or Lou Gerhig''s disease). Thus, this method may have applicability for detecting zinc-deficient SOD1 mutants in human ALS patients samples as well as in a transgenic mouse model of ALS and in cultured motor neurons. We determined previously that structural analogs of 1,10 phenanthroline, which react specifically with Cu(I), react with the active Cu(I) of SOD1 when zinc is absent, but not when zinc is also bound, as evidenced by the fact that the reaction is inhibited by pretreatment of the enzyme with zinc. We report herein that bathocuproine, or its water-soluble derivative bathocuproine disulfonate, react with zinc-deficient SOD1 to form a complex which fluoresces at 734 nm when excited at 482 nm. Fluorescent intensity is concentration dependent, thus we propose to use fluorescent confocal microscopy to measure intracellular levels of zinc-deficient SOD1 in situ. [Copyright &y& Elsevier]

Simple Summary: Oral squamous cell carcinoma (OSCC) accounts for 90–95% of malignant tumors of the lip and oral cavity and is associated with high mortality in the advanced stages. Early diagnosis is a challenge for oral pathologists and dentists, due to the ambiguous appearance of early OSCC, which is often misdiagnosed, mistreated, and associated with diagnostic delay. The gold standards for OSCC diagnosis are biopsy and histopathological assessment, but these procedures are invasive and time-consuming. Adjunctive noninvasive techniques allow the definition of the malignant features of a suspicious lesion in real time and noninvasively, thus improving the diagnostic procedure. The present review aimed to focus on some of the main promising noninvasive imaging techniques, to highlight their perspective adoption in a three-step diagnosis, which is idealistically faster and better, as well as enables the patient's compliance. Oral squamous cell carcinoma (OSCC) is the most prevalent form of cancer of lips and oral cavity, and its diagnostic delay, caused by misdiagnosis at the early stages, is responsible for high mortality ratios. Biopsy and histopathological assessment are the gold standards for OSCC diagnosis, but they are time-consuming, invasive, and do not always enable the patient's compliance, mainly in cases of follow-up with the need for more biopsies. The use of adjunctive noninvasive imaging techniques improves the diagnostic approach, making it faster and better accepted by patients. The present review aims to focus on the most consolidated diagnostic techniques, such as vital staining and tissue autofluorescence, and to report the potential role of some of the most promising innovative techniques, such as narrow-band imaging, high-frequency ultrasounds, optical coherence tomography, and in vivo confocal microscopy. According to their contribution to OSCC diagnosis, an ideal three-step diagnostic procedure is proposed, to make the diagnostic path faster, better, and more accurate. [ABSTRACT FROM AUTHOR]

Intracellular delivery of protein nanoparticles (NP) is required for nanomedicine. Our research was focused on the quantitative analysis of protein NP intracellular accumulation and biodegradation in dynamics along with host cytokine gene expression. Fluorescent NP fabricated by nanoprecipitation without cross-linking of bovine serum albumin (BSA) and human immunoglobulins (hIgG) pre-labeled with Rhodamine B were non-toxic for human cells. Similar gradual uptake of the NP during 2 days and subsequent slowdown until background values for 5 days for human cell lines and donor blood mononuclear cells revealed that NP internalization was neither cell-type nor protein-specific. NP delivery into cells was inhibited by homologous and heterologous NP but did not depend on the presence of BSA or hIgG in culture media. The protein NP internalization induced interferon α, β, λ but neither γ nor interleukin 4 and 6 gene expression. Accordingly, cellular uptake of non-toxic protein NP induced Th1 polarized innate response. Cellular uptake and biodegradation of albumin and immunoglobulin nanoparticles but not original proteins induce Th1 innate immunity. Unlabelled Image [ABSTRACT FROM AUTHOR]

Intracellular delivery of protein nanoparticles (NP) is required for nanomedicine. Our research was focused on the quantitative analysis of protein NP intracellular accumulation and biodegradation in dynamics along with host cytokine gene expression. Fluorescent NP fabricated by nanoprecipitation without cross-linking of bovine serum albumin (BSA) and human immunoglobulins (hIgG) pre-labeled with Rhodamine B were non-toxic for human cells. Similar gradual uptake of the NP during 2 days and subsequent slowdown until background values for 5 days for human cell lines and donor blood mononuclear cells revealed that NP internalization was neither cell-type nor protein-specific. NP delivery into cells was inhibited by homologous and heterologous NP but did not depend on the presence of BSA or hIgG in culture media. The protein NP internalization induced interferon α, β, λ but neither γ nor interleukin 4 and 6 gene expression. Accordingly, cellular uptake of non-toxic protein NP induced Th1 polarized innate response., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Influenza A virus (IAV) entry is a stepwise process regulated by viral and cellular cues, facilitating cellular functions. Virus entry begins by attachment of hemagglutinin to cell surface sialic acids, followed by endocytic uptake, vesicular transport along microtubules, low-pH-mediated viral membrane fusion with the late endosomal membrane, capsid uncoating, viral ribonucleoprotein (vRNP) release, and nuclear import of vRNPs. Here we show a basic methodology to visualize incoming and egressing IAV particles by correlative light and electron microscopy (CLEM). We combine fluorescence microscopy of virus-infected human lung carcinoma A549 cells with high-pressure freezing (HPF) and in-resin fluorescence CLEM and the Tokuyasu CLEM method. This approach forms a basis to study the virus life cycle and virus-host interactions at the ultrastructural level.

The gold standard of diagnosis for nonmelanoma and melanoma skin cancer has been skin biopsy with routine paraffin embedded hematoxylin and eosin histopathology. This practice is frequently carried out on suspicious lesions to rule out a malignant process. Therefore, as a result, many biopsies are done on benign lesions. Unlike other fields of medicine that rely on noninvasive imaging modalities, the use of imaging devices in dermatology has not been as robust. This has been mainly due to the limited resolution offered by imaging devices that is needed to detect malignant changes in the cutaneous layers. However, the demand for more efficient in vivo and ex vivo imaging tools to reduce the amount of biopsies have led to new areas of investigation using noninvasive modalities to augment the clinical diagnosis of skin cancer. The use of noninvasive imaging both in vivo and ex vivo has the potential to increase efficiency of diagnosis and management, decrease healthcare cost, improve clinical care and enhance patient satisfaction.

The antimicrobial peptide Ltc1-K and its derivates without one, two, then three N-terminal amino acid residues were studied based on the hypothesis (backed by some experimental data) that the hydrophobic N-terminal moiety of linear cationic antimicrobial peptides defines their haemolytic activity. It was discovered that the excision of three N-terminal amino acid residues considerably decreases the peptide's toxicity for eukaryotic cells and simultaneously increases the selectivity of antibacterial activity for some bacteria species. Studies performed with the model membrane systems and human erythrocytes revealed that the main reason for the observed effect is a multifold decrease in the peptide's affinity to an eukaryotic cellular membrane enriched with zwitterionic phospholipids.